[160_TD$IF]Opinion A Process of Resection- Dependent Nonhomologous End Joining Involving the Goddess Artemis Markus Löbrich1,* and Penny Jeggo2,* DNA double-strand breaks (DSBs) are a hazardous form of damage that can Trends potentially cause cell death or genomic rearrangements. In mammalian G1- and A c-NHEJ pathway has been defined G2-phase cells, DSBs are repaired with two-component kinetics. In both involving resection of DSB ends prior phases, a fast process uses canonical nonhomologous end joining (c-NHEJ) to their ligation in G1. Thus, the two to repair the majority of DSBs. In G2, slow repair occurs by homologous main pathways for repairing DSBs in G1 human cells are resection-inde- recombination. The slow repair process in G1 also involves c-NHEJ proteins pendent and resection-dependent c- but additionally requires the nuclease Artemis and DNA end resection. Here, we NHEJ. consider the nature of slow DSB repair in G1 and evaluate factors determining The resection process in G1 uses whether DSBs are repaired with fast or slow kinetics. We consider limitations in many of the same factors used for our current knowledge and present a speculative model for Artemis-dependent resection during homologous recom- bination in G2 but orchestrates them in c-NHEJ and the environment underlying its usage. a manner suited to a c-NHEJ process. Since Artemis is the only identified fac- tor involved in the resection process DNA DSBs Are Repaired by Multiple Pathways whose loss leads to unrepaired DSBs, The ability to repair DNA DSBs is a critical determinant of survival to ionising radiation (IR) and we refer to this process as Artemis- other DSB-inducing agents. However, the fidelity with which DSBs undergo repair is also and resection-dependent c-NHEJ. important because inaccurate rejoining can generate genomic rearrangements, potentially Loss of other resection factors pre- vents the initiation of resection but leading to carcinogenesis. Therefore, when evaluating DSB repair mechanisms, both rejoining allows resection-independent c- capacity and accuracy should be considered. NHEJ. Canonical nonhomologous end joining (c-NHEJ, see Glossary) is the predominant repair Artemis- and resection-dependent c- NHEJ makes a major contribution to pathway in all mammalian cell cycle phases for directly induced DSBs, such as those generated translocation formation and can lead to by IR. Homologous recombination (HR) contributes to the repair of such DSBs in S and G2 previously described microhomology- phases, although it primarily functions following replication fork stalling or collapse [1,2]. These mediated end joining. roles during replication likely represent the essential function of HR and its major contribution to maintaining genomic stability. An overview of the cell-cycle-dependent roles of c-NHEJ and HR is depicted (Figure 1A). Here, we focus on pathways that rejoin directly induced DSBs in G1 or 1Radiation Biology and DNA Repair, Darmstadt University of Technology, G2 phase. 64287 Darmstadt, Germany 2Genome Damage and Stability In addition to c-NHEJ and HR, which have the potential to rejoin DSBs accurately, there is Centre,[16_TD$IF] School of Life Sciences, University of Sussex, Brighton BN1 9 increasing recognition that there are also error-prone pathways involving enzymatically driven RQ, UK resection [3,4]. Alternative NHEJ (alt-NHEJ), which involves DNA ligases I or III, X-ray repair cross-complementing protein (XRCC)1, poly [ADP-ribose] polymerase (PARP)1 and DNA polymerase (Pol) u [3,5,6], represents one such pathway. It exploits microhomology (MH) *Correspondence: Microhomology-mediated end [email protected] during rejoining, thereby generating junctional deletions. (M. Löbrich) and joining (MMEJ) represents the rejoining of DSBs using small regions of MH [7] and is often [email protected] (P. Jeggo). 690 Trends in Biochemical Sciences, September 2017, Vol. 42, No. 9 http://dx.doi.org/10.1016/j.tibs.2017.06.011 © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Glossary Artemis: Protein whose deficiency leads to a form of radiosensitive severe combined immunodeficiency. It is often described as a c-NHEJ component, although it is only essential for a subfraction of DSB end joining. Artemis has nuclease activity, and a b-CASP and b-lactamase domain. Since it is hitherto the only identified factor whose loss leads to unrepaired DSBs specifically during resection- dependent c-NHEJ, we have called the process Artemis and resection dependent c-NHEJ. BRCA1/2: BRCA1 and 2 are frequently mutated in forms of hereditary breast cancer. BRCA2 functions in HR by aiding the loading of RAD51. The function of BRCA1 is less clear but it has roles during HR and Artemis-dependent c-NHEJ with the available evidence suggesting that it counteracts the anti-resection function of 53BP1. Canonical non-homologous end- joining (c-NHEJ): An end-joining DSB repair pathway that involves the initial binding of the Ku heterodimer to the DNA end. Then the DNA- dependent protein kinase catalytic subunit DNA-PKcs is recruited, generating the DNA-PK complex. Finally, a complex of DNA ligase IV, XRCC4, XLF, and PAXX carries out the rejoining step. Figure 1. Contribution of NHEJ and HR during the Mammalian Cell Cycle. (A) (i) c-NHEJ represents the major CtIP: Is the mammalian homologue repair pathway for two-ended DSBs in all cell cycle phases. (ii) HR repairs a sub-fraction of two-ended DSBs in G2 and late of the yeast protein Sae2 and is S phase, when a sister chromatid is present [12,79]. (iii) During S phase, HR also repairs one-ended DSBs that arise required for resection in mammalian following replication fork collapse [80]. The y axes for the panels on the right represent the relative usage of each pathway cells. It associates with the MRN throughout the cell cycle; it does not reflect the relative usage between the pathways. (B) Most X-ray-induced DSBs in G1 complex. and G2 are repaired with fast kinetics within the first 2–4 h in both cell cycles phases by c-NHEJ (red). However, a Microhomology-mediated end- subfraction of lesions is repaired with slower kinetics over many hours. This slow component requires ATM and ATM- joining (MMEJ): MMEJ is often dependent signalling proteins and is slower in G2 compared with G1 (compare the residual level of unrepaired DSBs at described as being synonymous with 1 day after X irradiation). Slow DSB repair in G2 is performed by HR (blue). In G1, the slow component represents a form of alt-NHEJ. However, most studies end joining involving resection and c-NHEJ proteins. This process also requires the nuclease Artemis. Hence, we have examining MMEJ do not assess the called it Artemis-dependent c-NHEJ. Since it has elements of c-NHEJ and HR, it is depicted in red with blue stripes. DSBs rejoining process. Here, we define induced by calcheomycin or neocarzinostatin are repaired with biphasic kinetics similar to X-ray-induced DSBs. In MMEJ as representing a form of contrast, following exposure to high linear[152_TD$IF] energy transfer (LET) a particles, the majority of DSBs are repaired with slow rejoining that exploits a short region kinetics in G1 and G2 (dashed lines) [8,13,38]. c-NHEJ, canonical nonhomologous end joining; DSB, double-strand break; of microhomology, which is HR, homologous recombination. inherently erroneous due to small deletions. However, since alt-NHEJ does not contribute significantly to DSB rejoining in G0/G1-phase taken as being synonymous with alt-NHEJ. However, most studies do not examine the human cells, we discuss here the underlying pathway. likelihood that most MMEJ in G0/G1- phase human cells occurs by Artemis-dependent c-NHEJ. Recent work has revealed another process of resection-dependent rejoining, namely resec- Homologous recombination (HR): tion-dependent c-NHEJ [8]. Here, we define MMEJ as DSB rejoining involving MH regardless of A DSB rejoining pathway that uses the pathway utilised and discuss the likely contribution of resection-dependent c-NHEJ and alt- extensive regions of homology and information from a homologous NHEJ to MMEJ later in the review. First, we discuss recent insight into the nature of resection- template. In mammalian cells, the dependent c-NHEJ and present a speculative model for its usage. Trends in Biochemical Sciences, September 2017, Vol. 42, No. 9 691 Insight Gained from Evaluating the Kinetics of DSB Repair sister chromatid provides the source Studies using pulsed-field gel electrophoresis (PFGE) or the neutral elution technique have of homology, therefore, HR only occurs in late S and G2 phase, after demonstrated thatDSBs arerepairedwithtwo-componentkinetics[9,10]. Enumeration ofgH2AX the sister chromatid has been foci in G0/G1 has confirmed that 80% of DSBs are repaired rapidly with the remainder being synthesised. The process involves rejoined with substantially slower kinetics [11] (Figure 1B). Both processes, however, require c- the generation of ss-DNA by end NHEJ proteins although the slow process additionally requires the nuclease Artemis [11]. Cell- resection, which is rapidly bound by fi RPA. RAD51 then replaces RPA by a cycle-speci c analyses have revealed that G2 cells show similar two-component kinetics, process involving BRCA2. This leads although the slow process in G2 is slower than its G1 counterpart (Figure 1B). Genetic analysis to strand exchange and the showed that in G2, while the majority of DSBs are repaired with fast kinetics by c-NHEJ, the slow formation of heteroduplex DNA. process represents HR [12]. But importantly in this context, the fast and slow repair processes in Finally, the region encompassing the DSB is recovered using the G0/G1 require c-NHEJ proteins but the slow process additionally requires Artemis.